Isolation,identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.     

Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin

A sulfated neuropeptide [pGlu-Ser-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2], with a blocked N-terminus and related to the undecapeptide leucosulfakinin, has been isolated from head extracts of the cockroach, Leucophaea maderae. It exhibits sequence homology with the hormonally-active portion of vertebrate hormones cholecystokinin, human gastrin II and caerulin. This peptide, termed leucosulfakinin-II, shares a common C-terminal heptapeptide fragment with leucosulfakinin and a comparison of the two sequences provides an assessment of the importance of the constituent amino acids to biological activity. Leucosulfakinin-II shows a greater resemblance to cholecystokinin than does leucosulfakinin. Leucosulfakinin-II and leucosulfakinin are the only two reported invertebrate sulfated neuropeptides. As with leucosulfakinin, the intestinal myotropic activity of leucosulfakinin-II is analogous to that of gastrin and cholecystokinin. The sequence homology between the leucosulfakinins and the vertebrate hormones, as well as their analogous myotropic activity, suggest that gastrin/cholecystokinin-like neuropeptides are not confined to vertebrates, but also occur in invertebrates.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Molecular forms of cholecystokinin in plasma from normal and gastrectomized human subjects following a fat meal

The present study was undertaken to characterize molecular forms of cholecystokinin (CCK) in human fat-stimulated plasma by Sephadex G50 column chromatography followed by radioimmunoassays employing 3 different region-specific antibodies. CCK was extracted and concentrated from plasma of healthy subjects by adsorption to SEP-PAK C18 cartridges and from plasma of gastrectomized patients by addition of 96% ethanol. Antibody 1703 binds to carboxy-terminal CCK-peptides containing at least 14 amino acid residues, antibody T204 to sulfated carboxy-terminal CCK-peptides and antibody 5135 to carboxy-terminal forms of CCK and gastrin. Four molecular forms of CCK were consistently demonstrated; peak I eluted in the void volume and comprised 1.8-10.2% of CCK-immunoreactivity, peak II eluted between the void volume and the CCK-33/39 standard and comprised 9.8-21.6%, peak III eluted at the position of the CCK-33/39 standard and comprised 42.4-55.4%, and peak IV eluted between the CCK-33/39 and CCK-14 standards and comprised 25.4-40.1% of CCK immunoreactivity. Since these 4 molecular forms reacted to all 3 CCK-antibodies it is likely that they contain the sulfated tyrosyl and carboxy-terminal regions of CCK and, therefore, possess biological activity.     

Signaling pathways mediating gastrin's growth-promoting effects.

In addition to its fundamental role in stimulating gastric acid secretion, the peptide hormone gastrin induces growth-promoting effects on diversity of target cells. Various mechanisms, including endocrine, paracrine, and autocrine, have been proposed for gastrin's growth-promoting actions. The mitogenic effects of gastrin are mediated by specific cell surface receptors activated after gastrin binding. The functionally defined receptors for gastrin include cholecystokinin A (CCKA) receptor, which is discriminating for sulfated CCK8; cholecystokinin B (CCKB)/gastrin receptor, which binds gastrin17 sulfated, and nonsulfated CCK8 with nearly equal affinities; cholecystokinin C (CCKC), which is a low-affinity gastrin binding protein; and novel, high-affinity receptors selective for amidated gastrin, processing intermediates of gastrin, or both. The signaling pathways mediating gastrin's stimulation of the CCKB/gastrin receptor have been progressively outlined, and the pathways mediating other receptors have been slowly emerging. Engagement of the gastrin receptor initiates various biochemical and molecular events, including recruitment and activation of tyrosine kinases, activation of the phospholipase C signaling pathway leading to phosphoinositide breakdown, intracellular calcium mobilization and protein kinase C stimulation, activation of the mitogen-activated protein kinase pathway, and induction of early response genes. Current emphasis is on understanding the functional significance of processing intermediate forms of gastrin, and the receptor subtypes and pathways that promote the trophic/mitogenic effects of the different molecular forms of gastrin.     

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.     

Receptor type for cholecystokinin on isolated intestinal muscle cells of the guinea pig

Smooth muscle cells isolated from the longitudinal muscle layer of guinea pig ileum were used to determine the presence and type of cholecystokinin/gastrin receptor mediating contraction. This was accomplished with a series of cholecystokinin and gastrin agonists (CCK-8, des(SO3)CCK-8, gastrin-17, des(SO3)gastrin-17 and pentagastrin) and antagonists (glutaramic acid derivatives CR 1392, CR 1409, CR 1505 and proglumide). The order of potency of agonists based on EC50 values derived from concentration-response curves was: CCK-8 greater than des(SO3)CCK-8 greater than gastrin-17 greater than des(SO3)gastrin-17. The inhibitory dissociation constant (Ki) for the antagonist CR 1505 derived from Schild plots was the same whether sulfated CCK-8 or desulfated gastrin-17 was used as agonist (4.47 +/- 0.76 versus 4.68 +/- 0.78 nM). Pentagastrin acted as a partial agonist and inhibited partially the response to CCK-8. The Ki values determined for all antagonists with pentagastrin as agonist were similar to those with CCK-8 as agonist. The order of potency of agonists and the independence of Ki values from the type of agonist used implied that CCK and gastrin interact with one receptor type; the receptor is more sensitive to CCK-8 but is minimally influenced by sulfation of the tyrosine residue. In this respect, the receptor appears to be distinct from the CCK receptor on gallbladder muscle cells and pancreatic acinar cells.     

Synthesis of analogues of the Des-Phe-NH2 C-terminal hexapeptide of cholecystokinin showing gastrin antagonist activity

Four analogues of Z-CCK-27-32-NH2, Z-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2, a cholecystokinin receptor antagonist have been synthesized by solution methodology. In these analogues, Z-Tyr(SO3-)-Nle-Gly-Trp-Met-Asp-NH2 16, Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-NH2 17, BOC-Tyr(SO3-)-Met-Gly-Trp-Met-Asp-NH2 24 and Boc-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp-NH2 25 methionyl residues were replaced by norleucyl residues. Preliminary biological activity on gastrin-induced acid secretion, in rat, are reported. These derivatives proved to antagonize the action of gastrin, with ED 50 of between 0.5 and 3 mg/kg.     

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.     

Identification of the site of sulfation of the fourth component of human complement

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.     

Sulphated CCK-8-like peptides in the neural ganglion of the protochordate Ciona intestinalis

Immunochemical studies were carried out on extracts of the neural ganglion from the ascidian Ciona intestinalis in order to the characterize the peptide(s), which react with antibodies against the C-terminal sequence common for the mammalian hormones, cholecystokinin (CCK) and gastrin. Radioimmunoassays specific for the sulphotyrosyl-containing N-terminus of CCK-8, for the common alpha-carboxyamidated C-terminus and for gastrin were used to monitor gel chromatography and reverse-phase HPLC of the extracts. Only neutral extracts contained immunoreactive material (634 (524-785) pmol eqv.CCK-8/g) (mean and range, n = 4)). HPLC revealed a small peak eluting almost like CCK-8 and a larger peak eluting earlier. By subsequent gel chromatography the larger peak eluted in the same position as sulphated CCK-8. The material was recognized almost equally by the N- and C-terminal CCK radioimmunoassays, whereas the specific C-terminal gastrin radioimmunoassay did not measure the peptides. Treatment with arylsulphatase removed the binding to the antiserum specific for the sulphotyrosyl-containing sequence of CCK. The results indicate that the ganglion of Ciona intestinalis contains a tyrosyl-sulphated peptide resembling mammalian CCK-8.     

Characterization of a tyrosine sulfotransferase in rat brain using cholecystokinin derivatives as acceptors

An apparently novel tyrosyl sulfotransferase activity was detected in a crude microsomal fraction from rat cerebral cortex by using 3'-phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) as the sulfate donor and various cholecystokinin (CCK) fragments or derivatives as acceptors. Among the latter, the shortest substrate was tert-butoxycarbonylaspartyltyrosine (Boc-Asp-Tyr), but the reaction was optimized by increasing the length of the peptide sequence on the C-terminal side up to tert-butoxycarbonylcholecystokinin octapeptide (Boc-CCK-8) as well as by the presence of acidic amino acid residues at the N-terminal side. Peptides with an N-terminal Tyr residue (e.g., CCK-7 or enkephalins) were not sulfated. With Boc-CCK-8 the optimum pH was 5.8, and apparent KM values were 0.14 +/- 0.02 mM for the peptide (0.5 microM PAPS) and 0.12 +/- 0.01 microM for PAPS (0.25 mM Boc-CCK-8). In the presence of 0.2 mM MnCl2 the Vmax of the reaction was enhanced without change of apparent affinities of the two substrates. The possible role of this sulfotransferase activity in posttranslational modification of CCK and other secretory proteins is suggested.     

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice

Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides.     

Inhibition of cell proliferation by the cholecystokinin antagonist L-364,718

Specific cholecystokinin (CCK) receptor and gastrin receptor antagonists were used to assess what role, if any, these receptors have in autocrine cell growth. Although the cholecystokinin receptor antagonist, L-364,718, inhibited cell proliferation in a broad spectrum of cell lines, the gastrin antagonist, L-365,260, had no effect on cell proliferation. In addition neither added gastrin17, nor sulfated cholecystokinin8, could reverse the inhibitory action of L-364,718. It is proposed that L-364,718 inhibits cell proliferation independently of classical gastrin/CCK receptors.     

Gastrin and cholecystokinin in the Eastern Grey kangaroo, Macropus giganteus giganteus

Gastrin and CCK are believed to have a common ancestor. The gastrin structure has probably evolved from CCK-like peptides at a stage later than the amphibians. To trace the evolution of gastrin and CCK we have determined their structures in an Australian marsupial, the Eastern Grey kangaroo. The brain CCK was identical to CCK-8 of most mammals. The larger form of kangaroo gastrin was a 33mer with the sequence pELHPQDLPHLMTDLSKKKGPWQEEDAAY(SO₃)GWMDF-NH₂. The 11 italic residues indicate differences from human gastrin. Gastrin-15 and gastrin-16 comprised about 70% of the total immunoreactivity and resulted from cleavage after the second and third residue, respectively, of the unusual tribasic cleavage site. The smaller forms of kangaroo gastrin differ from most other mammalian gastrins in that the N-termini are not blocked with a pyroGlu moiety. Unlike other gastrins, kangaroo gastrin is more than 95% sulfated. The present study indicates that the gastrin structure, as defined by having a Tyr at position 6 from the C-terminus, evolved before the marsupials diverged from the other mammals 130 million years ago.     

Tyrosine sulfation of arylsulfatase A and its peptide

Purified human liver arylsulfatase A (ASA) as well as an ASA peptide (residues 28-39) were sulfated by tyrosyl protein sulfotransferase in vitro. The media, but not the cell lysate, of normal human fibroblasts contained a tyrosine sulfated protein (pI = 4.5-5.5). This protein was not present in either media or cell lysate of human fibroblasts lacking ASA protein. These results suggest that tyrosine sulfation facilitates secretion of ASA and that this may have pathophysiological consequences.     

Molecular cloning of squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase and synthesis of a unique chondroitin sulfate containing E-D hybrid tetrasaccharide structure by the recombinant enzyme

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO(4)) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E-D hybrid tetrasaccharide structure.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.